ABSTRACT Proiect summary. Control of systemic angiogenesis in the lung remains pooriy understood despite the fact that neovascularization is an integral and pathologic feature of several lung diseases including chronic pulmonary thromboembolism. Ligating a pulmonary artery in experimental animals models the disease sequelae. We have shown that the innate immune system is critical for the growth of new vessels in mice after left pulmonary artery ligafion (LPAL) where resident macrophages release pro-angiogenic CXC chemokines. Yet, the process whereby neovascularizafion is stopped or slowed is unknown. Preliminary data demonstrate a role for the adaptive immune system in controlling the slow progression or cessation of neovascularization. We hypothesize that 1): CD4+ T-cells are recruited to the neovasculature and promote angiostasis. It is known that IL-10 is secreted by CD4+ T-cells, can limit infiammafion, and in our preliminary work, IL-10 deficient mice showed increased angiogenesis 21 days after LPAL compared to wild type LPAL mice. We further hypothesize that 2): angiostasis results from IL-10 release from CD4+ T-cells. Others have shown in vitro that IL-10 can significantly inhibit monocyte differenfiafion through downregulafion of granulocyte macrophage sfimulating factor and inhibit chemokine release from acfivated macrophages. Because we have shown that macrophage derived CXC chemokines appear to be critical for neovascularizafion and that the magnitude of angiogenesis 14 days after LPAL is closely correlated with the number of lung macrophages, we hypothesize that 3): IL-10 directly limits chemokine growth factor release from macrophages as the primary mechanism of angiostasis. Determining the molecular and cellular mechanisms involved in angiostasis is critical for the development of therapeutic strategies to limit chronic inflammation, prevent hemoptysis, and decrease pathologic systemic lung perfusion. Using our well-characterized mouse model of lung neovascularization during chronic pulmonary ischemia (>21 days after LPAL), we will determine the subtype of CD4+ T-cell that predominates in this chronic injury model and the mechanisms by which these cells limit lung angiogenesis. We will use flow cytometry, adoptive transfer, transgenic mice, and in vitro coculture, to define the role of CD4+ T-cell subtypes, their associated cytokines, and the mechanisms of angiostasis in the lung during chronic pulmonary ischemia. Programmatic interactions and synergy. The study of T-cell/macrophage interactions discussed in all three projects of this PPG, will provide a unique understanding and contrast ofthe hierarchy of immunologic processes contributing to different chronic lung (>21 days) pathologies. Project 3 specifically underscores the importance of a pro-inflammatory vasculature required for confinuous leukocyte recruitment during chronic inflammation, as shown in the preliminary results of Project 1. As in Project 2, we will determine the specific CD4+ T-cell subtype that predominates after the onset of pulmonary ischemia and use a variety of genetically modified mice provided by Dr. Powell (Project 2) where unique T-cell lineage pathways are blocked. In addifion to using similar methods for confirming CD4+ T-cells and cytokines (fiow cytometry, RT-PCR, ELISA, immunohistochemistry), we will study macrophage activation status in collaboration with Project 1 &2, and study physiologic functional changes in the chronically ischemic lung with Dr. Mitzner. Project 3 will make extensive use of both the Flow Cytometry Core as well as the Histology Core. The interacfion of three established investigators with years of research experience into the pathophysiology of complex lung disease, coupled with immunologist co-investigators who are experts in T-cell biology, macrophage activafion, and IL-10 function, provide an unparalleled opportunity to accomplish the aims of each of the three proposed projects.